Microscopic Observation Of Valsa Mali Var. Pyri Marked With GFP In Different Pear Tissues

Valsa canker is one of the main diseases of pear, which occurs in most pear cultivated areas in China, especially northern areas. In recent years, there is a gradually aggravated tendency. Preventive measures for pear Valsa canker often refere to those for apple Valsa canker, however there are some differences in colony morphology and host speciality. So far, there were more researches about the infection characteristics and pathogenesis of apple Valsa canker, but none report about pear Valsa canker in this areas. In this study, the pathogen of pear Valsa canker, V. mali var. pyri, was marked with GFP by using the agrobacterium-mediated transformation technique(ATMT). The transformations, which growth rate and biological characteristics were not significant changed comparing with their parent strains, were screened out. Using fluorescence microscopy, the processes of the infection and expansion of the transformations of virulent and hypovirulent strains in different pear tissues were observed, and the date in our study will offer references for the analysis of the infection characteristics and pathogenesis of pear Valsa canker. The results were as follows:1. Optimation of the Agrobacterium-mediated transformation system in V. mali var. pyri. V. mali var. pyri was marked with GFP through the agrobacterium-mediated transformation technique(ATMT). And the transformation efficiency under different conditions, such as different acetosyringone concentration, conidia concentration, co-cultivation time, co-cultivation temperature were compared. The results showed when the concentration of AS was 0.2 μg/m L, the conidia suspension was 1×105 /m L, cocultivation temperature 22 °C and cocultivation time 3 d, the efficiency of agrobacterium-mediumed transformation was optimum.2. Identification and screening of the transformations which were marked with GFP. 476 transformations of virulent strains(F-HN-6), and 237 transformations of hypovirulent strains(F-HN-2a-1) were obtained, of which 30 transformations of virulent and 23 transformations of hypovirulent strains were selected randomly to determine the stability of the fluorescence and hygromycin B resistance, and detect the hph and gfp gene by RT-PCR, to verify the transformations; And through the comparation of the growth rate, pathogenicity and colony morphology of wild type strain F-HN-6, F-HN-2a-1 and their transformations respectively, screening out the transformations of virulent strains F-HN-6-1, F-HN-6-3, F-HN-6-15 and the transformations of hypovirulent strains F-HN-2a-1-4, F-HN-2a-1-6, F-HN-2a-1-7, that characterizations were stable, and the growth rate and biological characteristics were not significantly changed from their parent strains.3. Observation of the infection and expansion of virulent and hypovirulent strains of V.mali var. pyri in pear organizations by Fluorescence Microscopy. Preparing the conidia suspension of virulent strains transformation F-HN-6-1 and hypovirulent strains transformation F-HN-2a-1-4 that had a certain concentration, then inoculate them on the healthy leaves and annual branches; Observing and comparing the infection and extension of virulent and hypovirulent strains transformation in pear organization. The results of Microscopic observation revealed that: There were differences between the transformations of virulent and hypovirulent strains in the infection and expansion in pear leaves and branches. Fungal hyphae expanded on the surface of upper epidermis through observing the infected leaves, which made the color of the leaves changed and formed a belt that tissue was necrotic but no hyphae colonization. The belts caused by transformations of virulent strains were wider than those by transformations of hypovirulent strains. The invasive ability of virulent strains was stronger than those of hypovirulent ones through observing the branches, hyphae of transformations, both from virulent and hypovirulent strains, existed mainly in the phloem and cortical tissue of the branch. There was no hypha in the epidermal tissue and a necrotic belt also formed in the front hyphae in the branch. The diseased tissue has dissociated.